Algae are increasingly being used as high density photobioreactors (Lee et al., “High density algal photobioreactors using light emitting diodes” Biotech. Bioengineering 44: 1161-1167 (1994)), in waste water treatments and elimination of heavy metals from contaminated water (Wilkinson “Mercury accumulation and volatilization in immobilized algal cell systems” Biotech. Letters 11: 861-864 (1989)), and for the production of useful products such as β-carotene (Yamaoka Seibutsu-Kogaku Kaishi 72: 111-114 (1994)) and pharmaceutical compounds. Unfortunately, most algae, especially eukaryotic algae, are not amenable to genetic manipulation for specific purposes. Many of the techniques that have been developed for the introduction of DNA into bacterial, yeast, insect, plant and animal cells are not optimal for algal systems, thereby limiting the usefulness of recombinant algae.
Algae, bacteria, and other microorganisms are particularly useful for making fermentation products that include organic acids, such as lactate, acetate, succinate, and butyrate, as well as neutral products, such as ethanol, butanol, acetone, and butanediol. Despite microorganisms producing ethanol, most fuel ethanol is currently still being produced from hexose sugars in corn starch or cane syrup utilizing organisms which metabolize these sugars into ethanol such as Saccharomyces cerevisiae or Zymomonas mobilis. However, these are relatively expensive sources of biomass sugars, often require fertilizers of fossil fuel origin, and have competing value as foods. Significantly, carbon dioxide is also generated as a by-product of fermentation. This is a property of all fermentative processes which is particularly undesirable as it contributes to atmospheric carbon dioxide.
Various attempts have been made to modify microorganisms to produce ethanol. The genes coding for alcohol dehydrogenase II and pyruvate decarboxylase in various organisms have been cloned and sequenced, and used to transform microorganisms to produce alcohols. For example, recombinant E. coli over-expressing Z. mobilis pyruvate decarboxylase were shown to have increased production of ethanol, although, very low ethanol concentrations were produced.
A superior approach is to link ethanogenesis to photosynthesis, utilizing sunlight as the main energy source, and carbon dioxide from the atmosphere as the main source of carbon for the synthesis of ethanol. Photosynthetic organisms do not normally express PDC or ADH, however, these genes have been introduced into a number of xenotypic organisms and have been shown to be fully expressed. For example, PDC and ADH genes of Z. mobilis have been cloned into a shuttle vector and used to transform the cyanobacterium Synechococcus. The PDC and ADH genes were expressed under control of the promoter of the cyanobacterial rbcLS operon which encodes the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. As a result of this process, ethanol accumulated in the culture medium, thereby demonstrating the principle that oxygenic photoautotrophic microorganisms can be genetically engineered to produce ethanol. U.S. Pat. No. 6,699,696 describes the genetic engineering of the photosynthetic Cyanobacterium Synechococcus sp. strain PCC 7942 to contain construct encoding the PDC and ADH enzymes from the Zymomonas mobilis pLOI295 plasmid as a method of producing ethanol. In another example, U.S. Application Publication No. 20030087368 describes the transformation of Rhodobacter with ethanogenic enzymes to produce ethanol. However, the use of photosynthetic prokaryotes to produce ethanol fails to generate quantities of ethanol significant or scalable with respect to energy requirements. Notably, this approach does not exploit the inherent efficiencies of enzyme chloroplast targeting.
The present invention relates to the creation and expression of a novel, genetically encoded cassettes coding for the sequestration of efficient ethanol producing systems in the chloroplasts of photosynthetic organisms, particularly eukaryotic algae, thereby producing ethanol in useful quantities.